The ability to culture human embryonic stem cells generated a great deal of excitement about their vast potential for treating a variety of human diseases such as diabetes, heart disease, neurological, retinal and skin disorders to name a few. Unlike adult stem cells, embryonic stem cells have indefinite growth potential and the potential to develop into any type of cell in the body, however, the precise conditions required for uniform differentiation into specific lineages remains largely unknown. Although, much research has been done to understand how to direct hESCs to develop into cells of a desired lineage, there currently is a critical need for methods of obtaining the well defined populations of progenitor or differentiated cells that will be needed to develop clinical grade materials for transplantation. The objective of this proposal is to identify cDNA encoded protein and peptide ligands that direct hESCs toward differentiation into cells of therapeutic interest. When hESC cells are grown as clusters of interacting cells known as embryoid bodies, they differentiate into cells of all 3 germ layers from which all body cells are derived. The specific aims of this proposal are to make cDNA display libraries from embryoid bodies and select them on differentiating hESCs to identify ligands that stimulate differentiation of hESCs. The selected ligands will be characterized to determine their specificity, biological activity and the type of progenitor cells that they interact with. The ligands will also be used to identify cognate receptors on progenitor cells for the production of progenitor binding antibodies. The resulting antibodies will be used to develop methods of obtaining well-defined progenitor populations for cell replacement therapy. Emphasis will be on but not limited to cardiac muscle, vasculature, and skin differentiation. The technology developed in this proposal will allow both directed differentiation of hESCs into desired cell types and the means to isolate well defined progenitor cell populations. The commercial products developed from this technology will include reagents that stimulate differentiation and reagents for cell isolation for use in clinical applications and research laboratories. There are considerable opportunities in the field of regenerative medicine to use embryonic stem cells to develop therapeutic products to treat diseases where healthy cells may be used to replace those lost to injury or disease. Embryonic stem cells have distinct advantages over adult stem cells like unlimited replication potential and ability to differentiate into virtually any cell in the body. However, currently there is a critical need to understand how embryonic stem cells differentiate and how to control the differentiation process to produce cells that are safe and suitable for therapy. This proposal is aimed at developing reagents and processes for controlled differentiation and purification of embryonic stem cells for therapeutic purposes. [unreadable] [unreadable] [unreadable]